Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species

INTRODUCTION: Invertebrate communities are central to many environmental monitoring programs. In freshwater ecosystems, aquatic macroinvertebrates are collected, identified and then used to infer ecosystem condition. Yet the key step of species identification is often not taken, as it requires a high level of taxonomic expertise, which is lacking in most organizations, or species cannot be identified as they are morphologically cryptic or represent little known groups. Identifying species using DNA sequences can overcome many of these issues; with the power of next generation sequencing (NGS), using DNA sequences for routine monitoring becomes feasible. RESULTS: In this study, we test if NGS can be used to identify species from field-collected samples in an important bioindicator group, the Chironomidae. We show that Cytochrome oxidase I (COI) and Cytochrome B (CytB) sequences provide accurate DNA barcodes for chironomid species. We then develop a NGS analysis pipeline to identifying species using megablast searches of high quality sequences generated using 454 pyrosequencing against comprehensive reference libraries of Sanger-sequenced voucher specimens. We find that 454 generated COI sequences successfully identified up to 96% of species in samples, but this increased up to 99% when combined with CytB sequences. Accurate identification depends on having at least five sequences for a species; below this level species not expected in samples were detected. Incorrect incorporation of some multiplex identifiers (MID’s) used to tag samples was a likely cause, and most errors could be detected when using MID tags on forward and reverse primers. We also found a strong quantitative relationship between the number of 454 sequences and individuals showing that it may be possible to estimate the abundance of species from 454 pyrosequencing data. CONCLUSIONS: Next generation sequencing using two genes was successful for identifying chironomid species. However, when detecting species from 454 pyrosequencing data sets it was critical to include known individuals for quality control and to establish thresholds for detecting species. The NGS approach developed here can lead to routine species-level diagnostic monitoring of aquatic ecosystems

Health Status of Sand Flathead (Platycephalus bassensis), Inhabiting an Industrialised and Urbanised Embayment, Port Phillip Bay, Victoria as Measured by Biomarkers of Exposure and Effects

Port Phillip Bay, Australia, is a large semi-closed bay with over four million people living in its catchment basin. The Bay receives waters from the Yarra River which drains the city of Melbourne, as well as receiving the discharges of sewage treatment plants and petrochemical and agricultural chemicals. A 1999 study demonstrated that fish inhabiting Port Phillip Bay showed signs of effects related to pollutant exposure despite pollution management practices having been implemented for over a decade. To assess the current health status of the fish inhabiting the Bay, a follow up survey was conducted in 2015. A suite of biomarkers of exposure and effects were measured to determine the health status of Port Phillip Bay sand flathead (Platycephalus bassensis), namely ethoxyresorufin-O-deethylase (EROD) activity, polycyclic aromatic hydrocarbons (PAH) biliary metabolites, carboxylesterase activity (CbE) and DNA damage (8-oxo-dG). The reduction in EROD activity in the present study suggests a decline in the presence of EROD activity-inducing chemicals within the Bay since the 1990s. Fish collected in the most industrialised/urbanised sites did not display higher PAH metabolite levels than those in less developed areas of the Bay. Ratios of PAH biliary metabolite types were used to indicate PAH contaminant origin. Ratios indicated fish collected at Corio Bay and Hobsons Bay were subjected to increased low molecular weight hydrocarbons of petrogenic origin, likely attributed to the close proximity of these sites to oil refineries, compared to PAH biliary metabolites in fish from Geelong Arm and Mordialloc.Quantification of DNA damage indicated a localised effect of exposure to pollutants, with a 10-fold higher DNA damage level in fish sampled from the industrial site of Corio Bay relative to the less developed site of Sorrento. Overall, integration of biomarkers by multivariate analysis indicated that the health of fish collected in industrialised areas was compromised, with biologically significant biomarkers of effects (LSI, CF and DNA damage) discriminating between individuals collected in industrialised areas from observations made in fish collected in less developed areas of the Bay

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

A Meta-Analysis Evaluating the Relationship between Aquatic Contaminants and Chironomid Larval Deformities in Laboratory Studies

Chironomid larval deformities have been widely investigated as an aquatic pollution toxicity end point. Field chironomid surveys often show a spatial association between contaminants and deformities, suggesting contaminants cause deformities. However, over 40 years of laboratory assays have not been able to confirm this causality. We therefore conducted a review of the literature and meta-analysis, in order to (A) assess whether trends across assays indicated dose–response effects, (B) characterize the consistency of results, and (C) investigate whether experimental issues and publication bias were contributing to inconsistency and/or reducing confidence in results. The experimental issues we investigated were extraneous nonchemical laboratory stressors (which may mask or interact with chemical effects), and mortality (which can confound deformity results). Our meta-analysis of the most commonly tested chemicals suggested dose–response effects for copper, but not lead or zinc. However, we also found substantial inconsistency across studies. Both mortality and extraneous stressors were potentially contributing to this inconsistency, reducing confidence in most published data. We observed no evidence of publication bias. We conclude that any causal link between contaminants and deformities remains uncertain, and suggest improved experimental and data reporting procedures to better assess this relationship

other_LC_metab_data

Concentrations of amine containing metabolites not involved in the cysteine metabolism of Chironomus tepperi deployed in microcosms. Detected by LCM

cyst_metab_LC data

Concentrations of amine containing metabolites involved in the cysteine metabolism of Chironomus tepperi deployed in microcosms. Detected by LCM

Chironomus_dryweight_raw

Dry weight data for deployed Chironomus tepper

Data from: Detecting copper toxicity in sediments: from the sub-individual level to the population level

1.Sediments accumulate chemicals that can be toxic to biota and often contribute to aquatic ecosystem decline. Measuring mortality in laboratory-bred organisms is a common way to assess sediment toxicity. However, mortality-based responses of resilient laboratory organisms may not reflect indigenous macroinvertebrate responses, which can be relatively more sensitive to sediment toxicants. A possible solution is to also measure responses at the sub-individual level. 2.Several organism responses to sediment copper toxicity were assessed in a field-based microcosm. Responses of laboratory-bred chironomids and snails deployed in microcosms were compared at sub-individual (metabolomic and gene expression), individual (survival and dry weight) and population (reproduction) levels, and contrasted to the abundance of colonizing macroinvertebrates in the microcosms. 3.Colonizing macroinvertebrate abundance showed a range of sensitivities based on EC50 (effect dose 50% change). Chironomidae made up 94.5% of the microcosm macroinvertebrates, with Paratanytarsus the most sensitive genus (EC50: 89 mg/kg copper) and Procladius the least sensitive (EC50: 681 mg/kg). 4.Survival of laboratory-bred organisms was the least sensitive response, comparable to decreased abundance of the least sensitive macroinvertebrate. Juvenile production in the snail, Potamopyrgus antipodarum, was the most sensitive population level response (EC50: 121 mg/kg), in contrast the snail Physella acuta was relatively more tolerant (EC50: 298 mg/kg). 5.Changes in sub-individual responses (gene expression and metabolite abundance) in laboratory-bred chironomid, Chironomus tepperi, were evident at 60 mg/kg. These changes likely reflect the direct effects of copper exposure and represent metal-specific responses. 6.Synthesis and applications. We showed that copper toxicity in sediments could be readily detected through changes in gene expression and metabolites in laboratory-bred chironomids exposed in field-based microcosms. These responses were more sensitive than mortality, and detected copper levels that caused microcosm chironomid populations to decline. These novel approaches will provide managers with new tools to better assess sediment toxicity